The present invention concerns novel G protein chimeras, nucleotide sequences encoding same, host cells transformed or transfected with same, methods of determining GPCR response to a molecule, and kits for same.
The guanine nucleotide-binding proteins (G proteins) are responsible for the efficient transmission of signals from agonist-bound cell surface receptors to different intracellular effectors. Approximately 5000 G protein-coupled receptors (GPCRs) are encoded by the human genome, and most fall into the categories of being Gq-, Gi-, or Gs-coupled. Some G proteins are more promiscuous than others by possessing the ability to interact with a large panel of GPCRs. The most notable examples of promiscuous G proteins are the human G16 and its murine homolog, G15. Both G15 and G16 link a variety of Gq-, Gi-, and Gs-coupled receptors to stimulate phospholipase C (PLC); (Offermanns, S. and Simon, M., 1995, J. Biol. Chem., 270; 15175-15180; Lee, J. W. M. et al., 1998, J Neurochem., 70: 2203-2211).
G proteins are membrane-associated proteins that transduce signals from GPCRs to various intracellular effectors. G proteins within this class are heterotrimers, consisting of an xcex1 subunit responsible for binding guanine nucleotides, a xcex2 subunit, and a xcex3 subunit. In mammalians, at least 16 distinct genes encode G protein xcex1 subunits. Furthermore, 5 distinct xcex2 subunit genes as well as 12 xcex3 subunit genes have been identified (Clapham, D. E. and Neer, E. J., 1997, Annu. Rev. Pharmacol. Toxicol. 37, 167-203; Hildebrandt, J. D., 1997, Biochem. Pharmacol., 51, 325-339). In vivo, the xcex2 and xcex3 subunits form high-affinity non-dissociating complexes, thus, a large number of xcex2xcex3 combinations are possible. Over the past decade, both the xcex1 subunit and xcex2xcex3 complexes have been shown to possess the ability to regulate effector systems (Clapham, D. E. and Neer, E. J., 1997, Annu. Rev. Pharmacol. Toxicol. 37, 167-203; Hildebrandt, J. D., 1997, Biochem. Pharmacol., 51, 325-339).
The G protein xcex1 subunit family is divided into four subgroups based on their amino acid sequence homology and functional diversity. The Gs family, including Gxcex1sL, Gxcex1sS and Gxcex1olf, is routinely classified as G protein xcex1 subunits able to mediate stimulatory regulation of adenylyl cyclase isoforms. The Gq family of xcex1 subunits, including Gxcex1q, Gxcex111, Gxcex114 and Gxcex116, promotes the activation of xcex2-isoforms of PLC. Gxcex112 and Gxcex113 are recently identified as the regulators of Na+-H+ exchangers and small molecular weight-G protein signaling cascades through the interaction with at least two guanine nucleotide exchange factors of Rho. The Gi family, which contains 10 membersxe2x80x94Gxcex1i1, Gxcex1i2, Gxcex1i3, Gxcex1o1, Gxcex1o2, Gxcex1o3, Gxcex1t1, Gxcex1t2, Gxcex1gust (Gxcex1t3), and Gxcex1zxe2x80x94was originally defined as the G protein xcex1 subunits closely related to those able to mediate inhibition of adenylyl cyclase (which is true of all Gxcex1i subtypes, as well as Gxcex1z). Gxcex1o subtypes mainly regulate calcium ion channels, while Gxcex1t, subtypes activate cGMP phosphodiesterases.
Because of their promiscuity, G15 and G16 have to date been recognised as being ideal candidates for linking xe2x80x9corphanxe2x80x9d receptors (cloned receptors without a known ligand) to PLC and its downstream effectors. Hence, G16 has received considerable attention as a potential tool for drug discovery (Milligan, G. et al., 1996, Trends in Pharmacol. Sci., 17: 235-237). Although G15 and G16 are more promiscuous than other G proteins, they are not true universal adapters for GPCRs. For example, the CCR2a chemokine receptor (Kuang, Y. et al., 1996, J. Biol. Chem., 271: 3975-3978), the xcex11A- and xcex11C-adrenoceptors (Wu, D. et al., 1992, J. Biol. Chem., 267: 25798-25802) are unable to recognize G16. Indeed, of thirty-three different GPCR examined to date (Offermanns, S. and Simon, M., 1995, supra; Lee, J. W. M. et al., 1998, supra; Kuang, Y. et al., 1996, supra; Wu, D. et al., 1992, supra; Wu, D. et al., 1993, Science, 261: 101-103; Zhu, X. and Birnbaumer, L., 1996, PNAS USA, 93: 2827-2831; Parmentier, M. L. et al., 1998, Mol. Pharmacol., 53: 778-786), at least six receptors are incapable of activating G16.
Most of the GPCRs that fail to activate G16 belong to the Gi-coupled receptor subfamily. The term Gi-coupled receptors stands for a group of seven transmembrane receptors that can interact with all three subtypes of Gxcex1i (Gxcex1i1-3)as well as Gxcex1z. Binding of a proper agonist to the receptor triggers the activation of the associated xcex1 subunits by promoting the release of GDP and the uptake of GTP. These receptors are widely distributed in different receptor categories, including aminergic, hormonal, peptidergic, purinergic, and chemokine. The Gi-coupled receptors constitute an exceedingly large GPCR subfamily which encompass many newly discovered receptors such as those for chemokines, however, approximately 15% of the Gi-coupled receptors examined to date cannot activate G16. As previously mentioned, this poses a serious concern for using G16 as an adapter of orphan receptors in drug screening protocols due to the large number of receptors which may not couple to or effectively couple to Gxcex116.
The underlying rationale for the intense interest in orphan GPCRs is based in their history of being excellent therapeutic targets. Over the past several decades, drug discovery programs world-wide have combined to produce greater than 200 novel drugs that possess activity or antagonizing properties towards GPCRs. As an example, it is estimated that the majority of drug discovery initiatives within the pharmaceutical industry are focused on this signalling pathway (Roush, W., 1996, Science, 271, 1056-1058). Likewise, the significance and complexity of GPCRs is readily apparent in the number of cases of genetic diseases that are known to be linked to various defects in these receptors (Dryja, T. P., et al., 1990, Nature, 343, 364-366; Sung, C-H. et al., 1991, Proc. Nal. Acad. Sci. U.S.A., 88, 6481-6485; Parma, J. et al., 1993, Nature, 365, 649-651; Shenker, A., et al., 1993, Nature, 365, 652-654; van den Oiweland, A. M., et al., 1992, Nat. Genet, 2, 99-102; Pan, Y., et al., 1992, Nat. Genet., 2, 103-106; Rosenthal, W. et al., 1993, J. Biol. Chem., 268, 13030-13033; Pollak, M. R., et al., 1993, Cell, 75, 1297-1303; Pollak, M. R., et al., 1994, Nat. Genet., 8, 303-307; Schipsni, E., et al., 1995, Science, 268, 98-100; Walston, J. et al., 1995, New Engl. J. Med., 333, 343-347; Widen, E., et al., 1995, New Engl. J. Med., 333, 348-351; Clement, K., et al., 1995, New Engl. J. Med., 333 352-354; Wajnrajch, M. P., et al., 1996, Nat. Genet., 12, 88-90; Clark, A. J. L., et al., 1993, Lancet, 341,461-462; Hager, J., et al., 1995, Nat Genet., 9, 299-304). As a result of the proven link of GPCRs to a wide variety of diseases and the historical success of drugs that target these receptors, characterisation of orphan GPCRs are among the most promising molecular targets for future drug discovery platforms. The ability to couple orphan GPCRs to down stream effectors via a small number of discriminating G proteins would greatly accelerate validation of GPCRs as potential drug targets and hence, further accelerate the discovery of novel therapeutics.
In light of the exceedingly large number of GPCRs, the characterisation of this class of proteins in toti is impractical, yet it remains necessary to identify ligands, particularly therapeutically effective ligands, for orphan receptors. Thus there is a need for an improved G protein with an increased promiscuity for binding to GPCRs (particularly Gi-coupled receptors) in order to link them to PLC and other downstream effectors. In theory, this would provide the means to identify useful ligands that bind to orphan receptors and result in the activation of an engineered G protein (possessing increased promiscuity) which would then allow the coupling of the receptor to a measurable downstream effector. If such ligands demonstrated high binding specificities towards a putative GPCR, they would become extremely useful research tools for delineating the receptor""s function and signal transduction pathway(s). Thus, identification of such ligands could play an important role in the validation of orphan receptors as viable drug targets, if such receptors were ultimately linked to discernible human disease states.
Several studies have indicated that distributed on the Gxcex1 subunit are multiple domains which confer specificity to GPCRs (Conklin, B. R. et al., 1993, Nature, 363: 274-276; Lee, C. H. et al., 1995, Mol. Pharmacol., 47: 218-223; Liu, J. et al., 1995, PNAS USA, 92: 11642-11646; Conklin, B. R. et al., 1996, Mol. Pharmacol., 50: 858-890; Kostenis, E. et al., 1997, J. Biol. Chem., 272: 23675-23681; Kostenis, E. et al., 1998, J. Biol. Chem., 273: 17886-17892). Modification of either amino or carboxyl-termini of Gxcex1 proteins has demonstrated the importance of such domains. The present invention, however, by using novel chimera Gxcex1 proteins, provides improved G proteins with greatly increased and enhanced promiscuity for GPCRs, particularly for Gi- and Gs-coupled receptors.
Experiments (below) have shown that Gxcex1 proteins having such substitutions have an increased promiscuity for GPCRs. Previously constructed chimeric Gxcex1 proteins (Chang, H. L. et al., 1995, Molecular Pharmacology, 47: 218-223), which included carboxyl-terminal substitutions, failed to increase G protein promiscuity and provided no suggestion as to how promiscuity might be improved. The difficulties experienced in improving G protein promiscuity are demonstrated by the large number of constructs made by the investigators which failed to improve promiscuity.
Thus according to the present invention there is provided a chimera comprising a Gxcex1 protein other than Gxcex1z having substituted at least one of the group of its carboxyl-terminal xcex2-sheet and/or xcex15-helix by that of Gxcex1Z.
Also provided according to the present invention there is a chimera comprising a Gxcex1 protein other than Gxcex1s having substituted at least one of the group of its carboxyl-terminal xcex2-sheet and/or xcex15-helix by that of Gxcex1s.
In particular, the Gxcex1 protein which is substituted may comprise Gxcex116.
Also provided are nucleotide sequences encoding chimeras of the present invention, host cells transformed or transfected with same, methods of determining GPCR response to a molecule, and kits for same.